Subcellular mechanisms in the action of adrenal steroids.

By in vitro experiments on rabbit bladder, we reassessed the traditional view that mammalian urinary bladder lacks ion transport mechanisms. Since the ratio of actual-to-nominal membrane area in folded epithelia is variable and hard to estimate, we normalized membrane properties to apical membrane capacitance rather than to nominal area (probably 1 μF ∼ 1 cm2 actual area). A new mounting technique that virtually eliminates edge damage yielded resistances up to 78,000 ΩμF for rabbit bladder, and resistances for amphibian skin and bladder much higher than those usually reported. This technique made it possible to observe a transport-related conductance pathway, and a close correlation between transepithelial conductance (G) and short-circuit current (I
sc) in these tight epithelia.G andI
sc were increased by mucosal (Na+) [I
sc∼0 when (Na+)∼0], aldosterone, serosal (HCO
 3
 −
 ) and high mucosal (H+); were decreased by amiloride, mucosal (Ca++), ouabain, metabolic inhibitors and serosal (H+); and were unaffected by (Cl−) and little affected by antidiuretic hormone (ADH). Physiological variation in the rabbits' dietary Na+ intake caused variations in bladderG andI
sc similar to those caused by the expectedin vivo changes in aldosterone levels. The relation betweenG andI
sc was the same whether defined by diet changes, natural variation among individual rabbits, or most of the above agents. A method was developed for separately resolving conductances of junctions, basolateral cell membrane, and apical cell membrane from thisG−I
sc relation. Net Na+ flux equalledI
sc. Net Cl− flux was zero on short circuit and equalled only 25% of net Na+ flux in open circuit. Bladder membrane fragments contained a Na+−K+-activated, ouabain-inhibited ATPase. The physiological significance of Na+ absorption against steep gradients in rabbit bladder may be to maintain kidney-generated ion gradients during bladder storage of urine, especially when the animal is Na+-depleted.

Na+ transport by rabbit urinary bladder, a tight epithelium

Isolated renal cortical collecting tubules obtained from rabbits treated chronically with desoxycorticosterone acetate (DOCA) have been found to possess elevated transepithelial potential differences and a greatly increased capacity for ion transport. Structural exmination of tubules from rabbits exposed to either DOCA or dexamethasone for 11--18 d reveals a marked increase in basolateral cell membrane area in these tubules. Morphometric analysis shows that this effect is specifically on the basolateral membrane area of only one of the two cell types found in this nephron segment. Increases of greater than 140% and 90% are found for the basolateral membrane area of the principal cells for DOCA and dexamethasone, respectively, but no change could be detected in the basolateral membrane area of the intercalated cells found in this nephron segment. No siginificant changes were found in luminal membrane area, cell number, or cell volume for either cell type. These observations demonstrate that significant changes in membrane area can occur in differentiated epithelia and suggest that this may be an important mechanism for modulating epithelial transport capacity.

https://rupress.org/jcb/article-pdf/81/2/439/1073646/439.pdf

Modulation of cell membrane area in renal collecting tubules by corticosteroid hormones

Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (PNa), intraepithelial Na activity (Nac), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N0), in the urinary bladder of the toad (Bufo marinus). To facilitate voltage-clamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85 mM) sucrose (50 mM) medium for the conventional Na-Ringer's solution on the serosal side. Aldosterone (5 X 10(-7) M, serosal side only) elicited proportionate increases in the Na-specific current (INa and in PNa, with no significant change in the dependence of PNa on mucosal Na (Nao). PNa and the control of PNa by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5 X 10(-3) M) which evoked coordinate and equivalent increases in INa and PNa. The aldosterone-dependent increase in PNa was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.

Aldosterone control of the density of sodium channels in the toad urinary bladder

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.

https://rupress.org/jcb/article-pdf/73/1/88/1072323/88.pdf

MM Civan

Papers

Effect of aldosterone on electrical resistance of toad bladder.